Chemical defense against predation in butterflies and moths has been studied since nineteenth century. A classical example is that of the larvae of the monarch butterfly Danaus plexippus, which feed on leaves of Asclepias curassavica (Asclepiadaceae), sequestering cardenolides. The adults are protected against predation by birds. Several other substances may be involved in chemical defense, such as iridoid glycosides, cyanogenic glycosides, glucosinolates, pyrrolizidine and tropane alkaloids, aristolochic acids, glycosidase inhibitors and pyrazines. The acquisition of these substances by lepidopterans can be due to sequestration from larval or adult host plants or de novo biosynthesis. Many Lepidoptera are known to be unpalatable, including the butterflies Troidini (Papilionidae), Pierinae (Pieridae), Eurytelinae, Melitaeinae, Danainae, Ithomiinae, Heliconiinae and Acraeinae (Nymphalidae), and Arctiidae moths, but knowledge of the chemical substances responsible for property is often scarce. This review discusses mainly three topics: field and laboratory observations on rejection of butterflies and moths by predators, correlation between unpalatability and chemicals found in these insects, and bioassays that test the activity of these chemicals against predators. Perspectives and future directions are suggested for this subject.

Chemical defense against predation in insects, particularly in Lepidoptera, is a well studied subject in chemical ecology with several reviews available1-10. As defined by Brower8, "chemical defense can be suggested when individual prey organisms contain one or more noxious chemical substances which facilitate proximal and/or distal rejectiona by predators; rejection can occur after a predator partially to completely ingests one or more prey individuals, or after the predator simply smells or tastes the prey".

The subject of chemical defense involves various areas of biology and chemistry. From a biological perspective, reports of prey rejection by predators have appeared since the nineteenth century. Bates11 and Müller12 were the first authors to propose that brightly colored butterflies were unpalatable to visually oriented predators, and that similarly conspicuous coloration in other palatableb or unpalatablec Lepidoptera evolved in order to enhance their protection through predator learning. Poulton13 pointed out that the unpalatability of butterflies was derived from their larval host plants. In the last 60-80 years chemical defense has been repeatedly tested against both vertebrate and invertebrate predators1,8,14-17. Evolutionary explanations for the reason why insects acquired noxious chemicals from host plants (so-called substances of secondary metabolism) began to take form after the seminal paper of Ehrlich & Raven18, who proposed a theory of "radiation and escape between plants and butterflies"d. In their scenario, three main steps promoted the diversification of both based mainly on evolution of protective chemicals in the plants: 1. plants with random mutations and recombinations could produce several chemical compounds not directly related to their basic metabolic pathways; 2. some of these compounds, by chance, would protect plants against attack by herbivores; the plants would then enter a new adaptive zone, promoting evolutionary radiation; 3. if insects had also random mutations and recombinations that enabled them to explore these new plant groups, selection would carry them into a new adaptive zone, where they would be free from competitors and natural enemies, promoting again an evolutionary radiation.

Chemical defense in insects involve several research areas and the investigations generally assume interdisciplinary feature. Exemplifying this multiplicity we can find studies on physiological mechanisms of biosynthesis and sequestration of defensive compounds by Lepidoptera21,22, evolution of warning coloration associated with unpalatability23-26 and techniques for isolation and identification of the defensive chemicals27.

The purpose of this review is to examine the progress in studies of secondary compounds thought to be involved in the chemical defense of Neotropical Lepidoptera. I organized it by classes of chemical compounds, focusing on three aspects: 1. field and laboratory observations on rejection of butterflies and moths by predators, 2. correlation between unpalatability and chemicals found in these insects, and 3. bioassays that test the activity of these chemicals against predators. Perspectives and directions for further research on the subject are suggested.

Chemical Compounds Acting as Defense in Neotropical Lepidoptera

Most organisms have alternate metabolic pathways in addition to those of primary metabolism that involve polysaccharides, lipids, proteins and nucleic acids. The natural products coming from such pathways are called "substances of secondary metabolism"28. In plants, from which butterflies and moths often sequester many of these substances, there are three principal building blocks for these compounds: 1. acetate, which via the mevalonate pathway leads to mono-, sesqui-, and diterpenes, iridoid glycosides and cardenolides; 2. amino acids, leading to cyanogenic glycosides, glucosinolates, pyrrolizidine alkaloids, tropane alkaloids and glycosidase inhibitors; and 3. shikimic acid, the precursor of many aromatic compounds such as furanocoumarins, aristolochic acids and b-carboline alkaloids (via aromatic amino acids). These substances take part in the chemical defenses in Lepidoptera and their roles will be discussed in detail in the next sections.

Iridoid glycosides

Iridoid glycosides29 (Figure 1, 1) are cyclopentenoid-monoterpene derived compounds in which the glycoside often occurs as an O-linked glycoside at C-1. They occur in about 57 plant families, and more than 600 iridoids structures have been described29.

These compounds have only been investigated in North American butterflies and moths. They are sequestered from their host plants by larvae of the nymphalid Euphydryas phaeton (host plants: Chelone glabra, Aureolaria flava¾ Scrophulariaceae and Plantago lanceolata¾ Plantaginaceae), E. chalcedona (Scrophularia californica¾ Scrophulariaceae), E. anicia (Besseya plantaginea, Castilleja integra¾ Scrophulariaceae), Poladryas arachne (Penstemon virgatus¾ Scrophulariaceae) and Junonia coenia (Plantago lanceolata), the pterophorid moth Ptatyptila pica (Castilleja sulphurea), the geometrid moth Meris alticola (Besseya plantaginea) and the sphingid moth Ceratomia catalpae (Catalpa bignonioides)10,29,30. Euphydryas and Poladryas retain the iridoids through the adult stages, while in the remaining species these compounds seem to be lost in the pupal stage10,29,30. Both adult and larva are warningly colored in Euphydryas, while in Junonia and Ceratomia larvae are conspicuous but the adults cryptic, suggesting that in the former both stages would be protected against predators, and in the latter only larvae would. Bowers and collaborators31-33 postulated that due to sequestration of iridoid glycosides from host plants the adults of the genus Euphydryas are generally unpalatable to birds. Bioassays with ants and spiders also demonstrated the role of iridoid glycosides in the chemical protection of larvae34-37.

In Neotropical environments Chai38 verified that adults of Thessalia ezra, a melitaeini butterfly that feed on Acanthaceae, was sight- and taste-rejected by birds, but no iridoid glycoside analyses were done. The investigation of all developmental stages of Thessalia and other butterflies that also feed on Acathaceae (e.g. Siproeta, Ortilia, Eresia and Anameca) and Plantaginaceae (e.g. Junonia) will be necessary to elucidate the role of iridoid glycoside in Neotropical species.

Cardenolides

Cardenolides or cardiac glycosides (Figure 1, 2) are, together with pyrrolizidine alkaloids, one of the best studied chemical defense system in insects, particularly in Lepidoptera39. The biosynthetic pathway of these compounds is not completely understood; cholesterol and b-sistosterol are metabolized in plants to pregnenolone, progesterone, and thence to cardenolides28. These compounds are found in 202 plant species in 55 genera and 12 Angiosperm families39.

The sequestration of cardenolides by North American Danaus and the rejection of these butterflies by birds have been studied for more than 40 years since the Browers40,41 showed that birds rejected the monarch butterfly D. plexippus. The presence of cardenolides in butterflies was shown to be highly effective against predation by Blue Jays (Cyanocitta cristata bromia, Corvidae). When fed with adults of D. plexippus reared as larvae upon a cardenolide plant, Asclepias curassavica (Asclepiadaceae), the birds exhibited typical effects of cardenolide poisoning, including repeated vomiting42. Monarchs reared on plants bearing cardenolides were much more emetic (= causing vomiting) than those reared on an asclepiad species lacking cardenolides41.

Some questions remain open about this system. For example, studies on the role of cardenolides in chemical protection of larvae against predators have received little attention. The presence of two kinds of chemical defense, cardenolides and pyrrolizidine alkaloids, in Danaus species44,45 is poorly explored from either a mechanistic or an evolutionary point of view. The dynamics of cardenolides in Neotropical species of Danaus need to be studied in relation to those found in the North American species.

Cyanogenic glycosides

Cyanogenic glycosides46 (Figure 2, 3-7) are O-b-glyco-sides of a-hydroxynitriles (cyanohydrins) biosynthetically derived from amino acids; these compounds have intermediate polarity and are water-soluble. They are accumulated in vacuoles in the plant and maybe to be so in animal cells. They generally co-occur with b-glycosidases and hydroxynitrile lyases, which are compartmentalized in other cells. The enzymatic cleavage of cyanogenic glycosides releases HCN plus sugar and ketones or aldehydes. The distribution of these compounds includes at least 2,650 plants (more than 550 genera and 130 families), with Passifloraceae as one of the main families. These compounds are also found in butterflies belonging to the Neotropical genera Heliconius (Nymphalidae, Heliconiinae), and Actinote, Altinote and Abananote (Nymphalidae, Acraeinae) 47,48.

Heliconius uses Passiflora species (Passifloraceae) as larval food plants47, and both larvae and adults biosynthesize de novo, from the amino acids valine and isoleucine, simple cyanogenic glycosides (linamarin and lotaustralin, 3 and 4, respectively ¾Figure 2)49. Passiflora species have a vast array of different cyanogenic glycosides, varying from simple aliphatic and aromatic compounds to sulphates and cyclopentenoid derivatives46,47 (Figure 2, 6 and7respectively). It has recently been demonstrated that a monoglycoside cyclopentenyl cyanogen was sequestered by Heliconius sara fed on Passiflora auriculata50. Moreover, it was found that H. sara has saurauriculatin (8), a thiol derivate from the cyclopentenoid cyanogenic glycoside epivolkenin (7), suggesting that the replacement of the nitrile group by a thiol would prevent cyanide release from the host plant50.

Into the neotropical acraeines, Brown and Francini48 showed that 16 species of Actinote, 12 of Altinote and one of Abananote may biosynthesize de novo these compounds in all developmental stages, since their larval host plants (mostly Eupatorium and Mikania, Asteraceae) do not have cyanogenic glycosides.

Heliconius species, together with Danaus (Nymphalidae: Danainae), are among the most studied species in relation to unpalatability. Several tests have demonstrated that they are unpalatable to vertebrate predators38,41,51-53. Chai38 verified that Actinote anteas and A. lapihta were sight-rejected by birds. However, there is much speculation in relation to the role of cyanogenic glycosides in chemical defense. The activity of these compounds against predators is poorly understood.

Glucosinolates

Glucosinolates (Figure 2, 9) are sulfur- and nitrogen-containing compounds biosynthesized through amino acid metabolism and are found mainly in the order Capparales (e.g. Cruciferae and Capparidaceae)54. Glucosinolates are known for their deterrent activity in plants against generalist herbivores and other natural enemies54. Their volatile derivatives are used as cues by specialist herbivores in the search of host plants and by parasitoids that attack insects feeding on glucosinolate-containing plants54. There are sparse data in the literature showing sequestration of glucosinolates by butterflies or moths from host plants and their role against predators.

Many Neotropical pierine butterflies (Appias, Ascia, Leptophobia, Itaballia, Pieriballia, Perrhybris)55,56 use Cruciferae and Capparidaceae as host plants, many of which may contain glucosinolates. Chai38,53 observed that the Neotropical Pierinae Melete, Appias, Perrhybris and Ascia were sight- and/or taste-rejected by birds. In experiments carried out in our laboratory it was verified that larvae of Ascia monuste, which feed on the crucifer Brassica oleracea, were taste-rejected by chicks. In both cases no chemical analyses were carried out to verify if glucosinolates were responsible for this activity.

Pyrrolizidine alkaloids

Pyrrolizidine alkaloids are probably the best studied defensive compounds in insects, especially in Lepidoptera. Many reviews on the activity of pyrrolizidine alkaloids in chemical defense and the role of these alkaloids in pheromone biosynthesis in Lepidoptera are available21,22,57-62.

Pyrrolizidine alkaloids are a diverse class of natural compounds based on a [3.3.0] azabicyclo ring, generally occurring as esters of a "necine base" with "necic acids" as mono- or diesters (Figure 3, 10-13)63. These alkaloids are known mainly from Asteraceae (tribes Eupatorieae and Senecioneae), Boraginaceae, Fabaceae (mainly in Crotalaria), Apocynaceae (subfamily Echitoideae, tribe Parsonsieae) and Orchidaceae (a few genera)21,60,63,65. They are postulated to occur in plants and Lepidoptera as N-oxides21,66, but recent work has discovered more polar metabolites in Ithomiinae butterflies67, similar to glycosylated pyrrolizidine alkaloids that have been characterized in Chrysomelidae bettles68.

Eisner14 was the first to point out the role of pyrrolizidine alkaloids as responsible for chemical defense of the arctiid moth Utetheisa ornatrix against the orb-weaving spider Nephila clavipes. Vasconcellos-Neto and Lewinsohn69 observed that the spider released, unharmed, Ithomiinae and Danainae butterflies from their webs. Brown15-17 found that pyrrolizidine alkaloids acquired from plants visited by adultse were responsible for this activity, since most Ithomiinae and Danainae do not fed as larvae on plants containing pyrrolizidine alkaloid. Other authors have shown the activity of pyrrolizidine alkaloids in other butterflies and moths against spiders74-78, lizards79 and birds79,80. Pure pyrrolizidine alkaloids were bioassayed against spiders81 and birds79,80; N-oxides were shown to be more active than free bases81-83. Corroborating the activity of pyrrolizidine alkaloids against predators, it is known that predators avoid or taste-reject danaine and ithomiine butterflies38,41,53. However, the role of glycosilated alkaloids against predators remains unknown.

Aristolochic acids

Aristolochic acids (Figure 4, 14) have been found only in plants belonging to the family Aristolochiaceae; biosynthetically, they are nitrophenanthrenes derived from aporphine alkaloids84. The unpalatability of these compounds has been postulated by several authors, but only one bioassay has been done with pure aristolochic acid, where the Japanese tree sparrow Passer montanus rejected rice grains treated with these compounds85,86. However, the authors pointed out that aristolochic acids alone have lower activity than that the total osmeterium secretion from the Asiatic Troidini Atrophaneura alcinous, which also contains sesquiterpenes and a complex mixture of more polar components, possibly sequestered from the host plant (Aristolochia debilis).

Rejection by birds of aposematic adult Troidini whose larvae feed on Aristolochia was described 35 years ago38,41,53 and aristolochic acids were found in several members of this tribe87-89. Chicks and ants also taste-rejected the aposematic larvae of the swallowtail butterfly Battus polydamas, but other invertebrate predators such as the reduviid bugs Arilus sp. and Montina confusa did not90. It is interesting to note that Aristolochia plants have other nitrophenanthrene derivatives, such as aristolactams (15) and benzoisoquinoline alkaloids (16)84, that have not yet been tested.

Glycosidase inhibitors

Glycosidase inhibitors are widespread in plants and can be sequestered by Lepidoptera, for whom they probably serve for defense by making the insects indigestible to a range of potential predators91,92. A very interesting case is reported for the uraniid Urania fulgens, a colorful, day-flying moth native to the tropical regions of Central America93. The larvae feed on Omphalea (Euphorbiaceae), particularly the liana O. diandra. Leaves of O. diandra contain polyhydroxypyrrolidine and a piperidine alkaloid analog (Figure 5, 17 and 18), sequestered by larvae and transferred to adults through the pupal stage; eggs also contained these alkaloids92. These azafuranose and azopyranose alkaloids, analog of hexose and heptose sugars, are potent inhibitors of glycosidases94. Adults of the ithomiine Mechanitis polymnia also show glycosidase inhibitors (polyhydroxylated nortropane alkaloids ¾ calystegines A3 and B2, 19 and 20)92,95, but their host plants (Solanum spp ¾ Solanaceae) were not analyzed. Although defensive functions have been proposed for these compounds, no bioassays have been carried out to show the activity of these substances against predators.

Pyrazines

Pyrazines are substances widespread in the plant and animal kingdoms and include some of the most powerful odors detected. The pyrazine nucleus comprises a six-membered aromatic ring containing two para-orientated tertiary nitrogen atoms96,97. Alkyl-substituted pyrazines are known to serve as trail-laying pheromones98 or alarm pheromes99 in some ants. In Lepidoptera, 2-methoxy-3-alkylpyrazines (Figure 6, 21) were found in several taxa of aposematic butterflies and moths, and sometimes in their larval host plants100,101. These substances potentiate the rejection response of rats and chickens when they drink an unpalatable quinine-water solution96,102,103. As suggested by these authors96,102,103, pyrazines might promote predation-learning of aposematic insects, since they have an extremely potent odor and a very low olfactory threshold. It is necessary to investigate the presence of pyrazines in other aposematic Lepidoptera (including all developmental stages) and compare them with cryptic ones. In addition, antipredator bioassays on pyrazines alone and together with other protective substances could give more information to draw a picture of the role of pyrazines in this context.

Other substances

Aposematic lycaenid larvae Eumaeus (Eumaeinae) are found in the Amazonian region feeding on Zamia sp. (Cycadaceae)104. It is known in North America that E. atala, whose larvae feed on the cycad Zamia floridana, is protected against ants (larvae) and birds (adults) by cycasin, a b-glycoside of methylazoxymethanol105 (Figure 6, 25).

The b-carboline alkaloids (Figure 6, 23 and24) are present in tissues of larvae and adults of Heliconius ismenius (Heliconiinae), and are sequestered from their larval host plant Passiflora costaricensis (Passifloraceae)106. The role of these alkaloids in chemical defense of Heliconius species is unknown.

Tropane alkaloids (Figure 4, 22) were found in aposematic larvae and adults of Placidula euryanassa and sequestered from the larval host plant Brugmansia suaveolens. The cryptic larva of Miraleria cymothoe, which feed on the same host plant excretes these alkaloids77. A biossay were carried out using Nephila spider as predator, but tropane were not active77. Further studies are needed to elucidate their role in the chemical defense.

Perspectives, future directions and caveats

Question on the antipredator role of secondary substances can be discussed in two main ways: "how" and "why" questions, similar to those discussed in animal behaviour107, and other biological areas. "How questions" can be summarized concerning the activity of the substances against predators and their action mechanisms. "Why questions" lead to evolutionary and ecological questions.

Focusing on "how questions" some problems remain to be solved in relation to chemical defense. The responses of predators to aposematic Neotropical butterflies and moths, behaviors such as liberation, rejection or non-attack are not always related to chemical compounds. Nevertheless, in some butterfly groups there is a close relationship between chemicals of larval or adult host plants and unpalatability. It is important to stress that correlation does do not mean a cause-effect relationship. It is necessary in most cases to isolate the chemicals from the insects and test them against natural predators. As examples, iridoid glycosides were tested only against spiders and ants, cardenolides against birds, pyrrolizidine alkaloids against ants, beetles, spiders, birds and lizards, but no bioassays with the other compounds were done. Other intriguing point is: are the substances per se or their metabolic and/or catabolic products responsible for antipredator activity? For example, cyanogenic glycosides are substances postulated to be unpalatable. These compounds are subject to metabolism by enzymes giving HCN, sugars and ketones or aldehydes44. The following questions rise from it: what compounds are really active against predators? Is there any synergistic interaction among them? For example, Petersen and collaborators108 showed that benzaldehyde is more active than HCN against ants but no bioassay with prunasin (Figure 2, 5), the parent compound, was done.

Another item concerning to "how questions" is the structure versus activity of chemical substances against predation. It is possible that chemical manipulation to extract and isolate these substances for bioassays produces non-natural by-products. Very recent examples of these are the characterization of N-oxides and glycosides of pyrrolizidine alkaloids, which have been found in plants and insects, respectively. Both were in past reports transformed to and isolated as free bases, using the usual chemical methodology: acid-base treatment, followed by reduction of N-oxides with Zn, may also hydrolyzed the glycosides. As stated above, N-oxides seems to be more active as free bases, but what is the role of the presumed glycosides of pyrrolizidine alkaloids? Studies on incorporation of pyrrolizidines into the integument of Neotropical Lepidoptera together with chemical defense activity of different pyrrolizidine chemical states (free bases, N-oxides, glycosides) must be done in order to better understand pyrrolizidine alkaloid activity against predation.

In relation to "why questions" we can formulate intriguing questions, some times very difficult to answer at light of the present knowledge. Are natural enemies the selective force responsible for the acquisition or biosynthesis of compounds by the prey? Or, are the substances sequestered or biosynthesized de novo due to other kinds of selective pressure, such as physiological restrictions? The recognition of substances by predators is not evidence of contact between them and prey containing these substances along of evolutionary time. As pointed out by Williams and collaborators109 if receptors are as conservative, as the genetic code or molecules such as histamine, the recognition of any molecules by them could be due to past interactions with ancient organisms such as microorganisms. Ecological relevance is easier to point out than evolutionary ones. For example, sympatric occurrence between prey and predators could be a signalization to ecological relevance. The best information would be the observation of predators releasing prey in the field and the utilization of those in a bioassay.

In addition to the relevant "how and why questions" the lack of knowledge of the natural history of a vast array of Neotropical butterflies and moths leaves us with a virtually unexplored field to study chemical defense. In groups such as Pierinae (e.g. Ascia and Melete)38,53 and Nymphalidae (e.g. Hamadryas, Diaethria, Callicore and Biblis)38,53,110-112 aversive response by predators was observed. Reports of rejection of skippers and other butterflies by the captive lion marmoset monkeys Leontopithecus rosalia (Callitrichidae) include Urbanus proteus and Astraptes creteus (Hesperiidae), and the nymphalids Caligo beltrao (Brassolinae), Morpho spp. (Morphinae) and Nica flavilla (Biblidinae)113. Data of Collins and Watson114 on field observations of bird predation on Neotropical moths suggest that the Geometridae are more unpalatable than Arctiidae, being the late a classical case of aposematic moths.. The causes and chemicals involved in the unpalatability of these groups have not been studied.

Finally, studies of chemical defense in Lepidoptera were done using mainly adults, but there is evidence that chemical defensive strategies may differ between the two actively feeding developmental stages of Lepidoptera (larvae and adults). As larvae suffer the constraints of single host plant and relative immobility they might have a wider array of defensive strategies than the free feeding and mobile adults. Unpalatable larvae have several mechanisms such as (1) stinging or irritating hairs or spines, (2) osmeteria and other eversible glands, (3) regurgitation, (4) presence of toxic leaf material in the gut, and (5) sequestration of chemicals from the host plant or de novo biosynthesis10. For example, fatty acids and sesquiterpenes, sometimes liberated in the hairs of larvae of Dione juno and Abananote hylonome are active against ants115; compounds whose biogenesis is unknown. Sequestered compounds can also be lost in the change from larvae to pupa, due to the metabolic cost to handle them9-10. Therefore, larvae could use a different set of chemicals, or different defensive strategies from those of adult.

The items pointed above presented some problems involving the role of secondary substances in the chemical protection of Neotropical butterflies and moths. The investigation of these topics, here directed at Neotropical Lepidoptera (these comments can also be addressed to aposematic insects in general), will rise with the increase of studies in this area, can help us to understand "how and why chemical substances are used by insects".

aProximal rejection involves contact with the prey in order to taste or smell it, while in distal rejection the predator perceives the prey from a distance due to odor cues, avoiding physical contact. In the later case, visual or acoustic cues are involved in mimicry systems.

c Müllerian mimicry: similarity in appearance of one species of animal to that of another, where both are unpalatable to predators. Both gain from having the same warning coloration, since the predator learns to avoid both species after tasting either one or the other.

d For a criticism on this theory see Futuyma and Keese 19 and Schoonhoven and coworkers 20 , and references therein.